3.2.1 Single Oligonucleotide Mutagenesis and cloning Approach (SOMA)
For determining PHD-2 morpholino specificity, the SOMA method was employed (Pfirrmann et al., 2013). A construct was made, in which the PHD-2 MO target sequence was inserted at the 5’- end of the GFP sequence contained in the vector pTP 218. For the construction a 74 bp long primer was designed consisting of a mutated EcoRI sequence (GAATTC - CAATTC), PHD-2 WT sequence complimentary to PHD-2 MO (red) and a start codon (ATG) after PHD-2 MO target sequence for the translation of following GFP sequence (green).
Primers were phosphorylated at the 5’ end. In a 50 µl reaction, 2 µl 100 µM primers, 5 µl 10x T4 polynucleotide kinase buffer, 0.5 µl 100 mM ATP and 1 µl T4 polynucleotide kinase were added to 41.5 µl dH2O. The reaction mixture was incubated at 37°C for 1 h. To stop the reaction, the mixture was incubated at 65°C for 15 min.
A PCR reaction was carried out in a final volume of 50 µl. To 23.5 µl dH2O, 2.5 µl phosphorylated primer (100 µM), 10 µl NAD+ (5 mM), 10 µl 5x HF buffer, 1 µl Phusion polymerase, 1 µl Taq DNA ligase, 1 µl dNTP mix (10 mM) and 1 µl template (100 ng) were added, mixed. For the PCR reaction the following cycle conditions were used. Initial denaturation of the DNA template at 95°C for 1 min, followed by 30 cycles of DNA denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 4 min, and a final extension at 72°C for 10 min. The template DNA in the PCR mixture was digested for 2 h at 37°C by the addition of 3 µl DpnI (Fast digest; Fermentas) and 5 µl 10x Fast digest buffer. Finally, the PCR product was transformed into E.coli and colonies were selected on ampicillin containing LB agar plate. For the mutated PHD-2 construct, a primer containing multiple point mutations was designed
3.2.2 Preparation of electro-competent bacteria
A single colony of E. coli XL-1 Blue was picked from a LB plate containing tetracycline, inoculated in 3 ml LB medium without antibiotics, and cultured overnight at 37°C with a rotary speed of 220 rpm. This 3 ml bacteria culture was then added to 300 ml LB medium without antibiotics in a 1 l flask, and cultured at 37°C with a rotary speed of 220 rpm for about 3 hrs until the OD 600 reached approximately 0.5. The culture was cooled on ice. Meanwhile, the centrifuge cups, pipets and 1.5 ml eppendorf tubes required for the preparation were all pre-cooled at 4°C. The bacteria were transferred to a pre-pre-cooled centrifuge cup and precipitated by centrifugation at 6,000 rpm for 15 min at 4°C. The supernatant was discarded. The pellet was gently re-suspended in ice cold 10% glycerol (autoclaved) and collected again by centrifugation at 6,000 rpm for 20 min. The washing step with the chilled 10% glycerol was repeated three more times and the pellet was finally re-suspended in 2 ml 10% glycerol. The bacteria were aliquoted in 50 μl per eppendorf tube on ice and immediately frozen in liquid Nitrogen. Aliquots were stored at -80°C.
3.2.3 Electroporation
2 μl ligated plasmid was added to 50 μl electro-competent bacteria (thawed on ice) and gently mixed by tapping. After incubation on ice for 5 min, the cell-DNA mixture was transferred to a chilled 1 mm electroporation cuvette (Equibio, UK) and transferred to an electroporator (Electro Square PoratorTM ECM830, BTX). The sample was pulsed once (500 V for 8 msec) and added 600 μl chilled LB medium to it. After being gently mixed by pipetting, the bacteria were kept on
5’
ggatcccatcgattccaattcataattgggagacagcagacagacataATGtctaaaggtgaagaattattcac 3’PHD-2 target GFP
Mutated EcoRI
Insert Vector
Vector
Page | 26 3 Methods ice. This 650 μl bacteria was transferred to a 1.5 ml eppendorf tube and incubated at 37°C for 30 min. A 50 μl aliquot and the rest were spread on two LB-Amp plates respectively, and incubated overnight at 37°C.
3.2.4 Colony PCR
A single colony was picked with an autoclaved toothpick from a LB-Amp plate and scratched on a fresh LB-Amp plate. The rest of the bacteria on the toothpick were rinsed in 10 μl ddH2O.
This 10 μl bacteria suspension was heated at 95°C for 10 min to lyse the bacteria, and 8 μl of it was used as the template for the colony PCR. A standard 25 μl colony PCR reaction contained 8 μl of the template, 1.5 mM MgCl2, 2.5 μl Taq polymerase buffer (supplied with enzyme, without MgCl2), 1 μM each of a forward primer and a reverse primer, 0.1 mM dNTPs and 0.1 μl Taq polymerase (5 u/μl, Fermentas). The PCR reaction was run using a thermocycle program with activating the enzyme and denaturing the DNA template at 95°C for 2min, followed by 26 to 30 cycles of DNA denaturation at 95°C for 45 sec, annealing at 55-58°C for 45 sec and extension at 72°C for 45 sec to 2 min according to the length of the PCR product (1kb/1min as recommended by the manufacturer), and the final extension at 72°C for 10 min. The PCR products were analysed on a 1% agarose gel along with 1kb DNA Ladder (Fermentas).
3.2.5 Plasmid mini-preparation
Bacteria were grown in 3 ml LB medium containing appropriate antibiotics overnight at 37°C.
1.5 ml of the bacterial culture was collected in an eppendorf tube and centrifuged at full speed for 30 sec in a bench top centrifuge. The supernatant was removed. The pellet was fully re-suspended in 100 μl of solution A and incubated on ice for 5 min. 200 μl of solution B was added, mixed and incubated on ice for 5 min and finally 150 μl of solution C was added and mixed. After incubation on ice for 5 min, the bacterial lysate was centrifuged at full speed for 10 min at RT. 400 μl supernatant was put in a fresh 1.5 ml eppendorf tube and plasmid DNA was precipitated by adding 1 ml absolute ethanol. After 5 min incubation at RT, plasmid DNA was pelleted by centrifugation for 3 min at full speed. Ethanol was discarded and the pellet was washed with 500 μl 70% ethanol. After discarding ethanol, the pellet was air dried and dissolved in 50 μl TE buffer (pH 7.5) containing RNase A (50 μg RNase A per ml TE). 1 μl was taken for restriction analysis (Sambrook et.al., 1989).
3.2.6 Plasmid maxi-preparation
When using the plasmid DNA for quantitatively and qualitatively demanding applications such as in vitro transcription, plasmid was extracted using maxi-preparation protocol.
Bacteria were grown in 200 ml LB medium containing appropriate antibiotics overnight at 37°C in a shaker. All the bacterial culture was collected in a 200 ml centrifuge tube and centrifuged at 5000 rpm (Sorvall) for 10 min. The supernatant was discarded and the pellet was re-suspended in 50 ml of solution A and transferred into 50 ml falcon tube. Bacteria was lysed by adding 50 μl Lysozyme (10 mg/ml), mixed and incubated on ice for 10 min. To this, 20 ml of fresh solution B was added, mixed and incubated on ice for 10 min and finally 15 ml of cold solution C was added and mixed. After incubation on ice for 10 min, the bacterial lysate was centrifuged at 5000 rpm for 15 min at 4°C. The lysate was filtered into a fresh 50 ml falcon tube. To the filtered supernatant, 20 ml isopropanol was added, mixed, incubated for 15 min at RT and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was discarded, the pellet was dissolved in 4 ml dH2O plus 4 ml 5 M LiCl and incubated on ice for 20 min. The extract was then spun for 10 min at 4000 rom at 4°C and the supernatant was transferred to a 30ml Corex tube. Plasmid DNA was precipitated by adding 18 ml absolute ethanol and incubated at -20°C for 1 h. Plasmid DNA was pelleted by centrifugation for 10 min at 9000 rpm, 4°C. The supernatant was discarded, the pellet was dissolved in 500 μl TE buffer (pH 7.5) and transferred to a 1.5 ml eppendorf tube. 10 μl RNase A (10 mg/ml) was added and incubated at 60°C for 30 min. Then 10 μl Proteinase K (10 mg/ml) was added and incubated at 37°C for 1 h. The plasmid DNA was extracted using phenol/chloroform. To the extract, 50 μl 3 M sodium acetate (pH 5,2) and 1 ml 100% EtOH was added and incubated overnight at -20°C. On the next day, it was spun at highest speed for 10 min at 4°C and the pellet was washed with 70% EtOH. The pellet was then air dried and dissolved in 200 μl 1x TE (pH 7.5) containing RNase A (50 μg RNase A per ml TE). 1 μl was taken for restriction analysis (Sambrook et.al., 1989).
3.2.7 In vitro synthesis of sense RNAs
To prepare synthetic capped RNA, the SP6 mMessage-mMachine™ Kit (Ambion) was used according to the manufacturer's protocol. A 20 μl reaction contains 1-1.5 μg linearized plasmid template, 2 μl 10x reaction buffer, 10 μl 2x NTPs, 2 μl enzyme mix. Transcription was carried out at 37°C for 2.5 hrs. The DNA template was removed by addition of 2 U DNaseI followed by incubation at 37°C for 30 min. The mRNA was purified with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and eluted with 20 μl RNase-free H2O. The concentration of synthesized RNA was determined using the NanoDrop® Spectrophotometer ND-1000 (peQlab, Germany), and the quality was examined on a 1% agarose gel. The synthesized RNA was stored in aliquots at -20°C.
3.2.8 In vitro synthesis of anti-sense RNAs
The preparation of digoxigenin/fluorescein labelled antisense RNA was carried out in a 20 μl reaction mixture containing 1-1.5 μg linearized template plasmid, 4 μl 5x Transcription buffer
Page | 28 3 Methods (Fermentas), 2 μl 0.1 M DTT, 0.5 μl RNase OUT (Invitrogen), 1 μl RNA polymerase (Fermentas), and 4 μl Digoxigenin/fluorescein mix (a mix of 10 mM ATP, 10 mM GTP, 10 mM CTP, 6.5 mM UTP, and 3.5 mM Dig-11-UTP/fluorescein, Roche). The reaction mixture was incubated at 37°C for 2.5 hrs, and the DNA template was removed by addition of 2 μl DNaseI (Fermentas) and the following incubation at 37°C for 30 min. Antisense RNA probes were purified by adding 300 μl 96% ethanol, 33 μl 7.5M ammonium acetate and 100 μl ddH2O to the mixture. The mixture was incubated for 30 min or overnight at -80°C and centrifuged at 4°C at 15000 rpm for 15 min. The pellet was rehydrated in 100 μl ddH2O. The concentration was measured using “Nanodrop”. The purified RNA probe was diluted in hybridization mix at a concentration of 1µg/ml and stored at -20°C.
3.2.9 Extraction of the total RNA from staged embryos
10 embryos were collected in a 1.5 ml eppendorf tube and immersed with 500 μl Trizol.
Embryos were then completely homogenized by sequentially passing 10 or more times each through needles with 0.9, 0.7, and 0.4 mm diameter fitted to an RNase-free syringe. To the embryo lysate, 100 μl chloroform was added and the two-phase mix was vortexed briefly. The lysate was then centrifuged at 10000 rpm at 4°C for 5 min. The upper phase was transferred to a new tube and re-extracted with an equal volume of chloroform, vortexed, spun at 10000 rpm at 4°C for 2 min. The supernatant was transferred to a new tube, mixed with 500 μl isopropanol, vortexed briefly. The precipitated RNA was isolated by centrifugation at 10000 rpm at 4°C for 5 min. The RNA pellet was washed with 500 μl 70% ethanol and air-dried. The pellet was re-suspended in RNase free H2O (50-100 μl).
3.2.10 Semi-quantitative polymerase chain reactions (SQ-PCR)
The first strand cDNA was synthesized with the Super Script™ II Reverse Transcriptase Kit (Invitrogen) according to the manufacturer’s protocol. 500 ng of total RNA from staged embryos was mixed with 2.5 μl random hexamer primer (0.2 μg/μl), 2.5 μl dNTP mix (10 μM) and filled with H2O to a volume of 30.5 μl. After gently mixing and a brief centrifugation step, the mixture was incubated at 65°C for 5 min. Then the mixture was chilled on ice. To the mixture were further added 10 μl 5x transcription buffer, 5 μl 0.1M DTT and 2 μl Ribonuclease Inhibitor (20 u/μl). The mixture was gently mixed and and incubated at 25°C for 2 min. Finally, 2μl Reverse Transcriptase (200 u/μl) was added and mixed. This reaction mixture with a final volume of 50 μl was incubated at 25°C for 10 min followed by 42°C for 50 min and in the end heating to 70°C for 10 min to stop the reaction. A standard 20 μl PCR reaction contained 1 μl cDNA obtained from RT reaction, 2 μl of 10x reaction buffer, 1 μl of 10 µM dNTP mix, 0.5 μl of specific primer mixture (forward and reverse primers, 7.5 μM for each), 0.3 μl Taq polymerase (5 u/μl, Fermentas), and 14.7 μl ddH2O. The PCR program used was as follows:
pre-denaturation at 95 ºC for 10 min, 26 cycles (for ODC-1) or 28 cycles (for HIF-1α eoe1 and VHL eoe2) of denaturation at 94ºC for 1 min, annealing at 58ºC (for HIF-1α eoe1) or 56ºC (ODC-1) or 50ºC (VHL eoe2) for 30 sec and extension at 72 ºC for 1 min, followed by final extension at 72 ºC for 10 min. 10 µl of the reaction mix was taken out for analysis. The PCR products were separated on a 1 % agarose gel and photographed with Bio-Rad Gel Doc 2000 (Bio-Rad, USA).
3.3 Handling and manipulation of Xenopus embryos